Molecular tools for the specific and rapid detection of toxigenic fungal contaminants in harvested commodities

Among natural contaminants, mycotoxins represent a major issue in food safety. Mycotoxins are secondary metabolites produced by fungi belonging to the genera Aspergillus, Alternaria, Penicillium, and Fusarium. Fungal contamination begins during crop cultivation and continues throughout postharvest handling and transformation phases. Therefore, early detection and monitoring are essential to prevent irreversible health impacts in humans and livestock. Besides the necessity to be highly specific, sensitive, accurate, and reliable, detection techniques need to be fast, easy to use, cost-effective, able to detect pathogens even in complex matrices, and ideally easy to deploy on sites. In the present work, two detection assays were developed: a multiplex TaqMan real-time PCR assay for the rapid and sensitive screening of multiple infections and LAMP assay for the onsite detection of Alternaria alternata. TaqMan assay targeted distinct mycotoxigenic fungi: A. alternata, Penicillium expansum, Aspergillus ochraceus, and Aspergillus carbonarius. Sets of primers and probes were designed on genes involved in mycotoxin production: pksI (alternariol), idh (patulin), and otaY (ochratoxin A) genes. A specific multiplex to detect different deoxynivalenol chemotypes of Fusarium culmorum was also set up. Specificity of the primers/probes was confirmed using genomic DNA from producing and non-producing species. Sensitivity, repeatability, and reproducibility of the assay were evaluated using artificially inoculated matrices. Instead, the LAMP assay was designed for both colorimetric and fluorometric applications and validated on artificially inoculated and naturally contaminated food matrices to determine its sensitivity and reproducibility. Both applications were proven to be highly sensitive and specific for detecting A. alternata in various food matrices even when using unpurified DNA. The application of the detection assays to be applied designed in this study would contribute to food safety by improving the prediction of the different sources of contamination in harvested commodities at both laboratory and point-of-care level.